The overall goal is to understand how sound is coupled to individual stereocilia within hair bundles of mammalian cochlea. Previous experiments have been limited by poor stimulus-probe coupling to bundles, which produces stimuli that are inhomogeneous in time and amplitude across stereocilia. Here, I propose to use silicon microfabrication and carbon nanotube technologies, together with new nanotube chemistries, to develop custom-fitted cochlear stimulus probes. These probes will be used to investigate stereocilia bundle mechanics, and (in subsequent projects) the micromechanics of transduction in cochlear hair cells. The project will also create a resource for the cochlear physiology community. In one aim, the anatomical dimensions of the V-shaped inner and outer hair cell hair bundles in the mouse cochlea will be quantified by labeling their membranes with FM1- 43 and visualizing them using confocal microscopy. Using these measurements a silicon based V-shaped stimulation probe will be microfabricated and its tip will be coated with carbon nanotubes (CNTs) to promote adhesion of the probe to the cochlear bundles. Adhesion will be tested in FM-143 labeled mouse cochlear explants by attaching the probe to the bundle, displacing the probe and imaging the motion of the bundle under a confocal microscope. If necessary, CNTs will be coated with glycoproteins or stereociliaspecific antibodies to promote adhesion. In the second aim, the V-shaped stimulus probe will be used to quantify the displacement of individual stereocilia within the inner and outer cochlear hair bundles in the mouse cochlea. The probe will be driven by a piezoelectric actuator using varying frequency sinusoids. The motion will be observed using a two photon microscope and DIC optics and quantified by applying Fourier analysis on the captured images. The same experiments will be repeated after applying the calcium chelator BAPTA to cut the tip links. The resulting measurements will help us understand the contribution of the tip links to hair bundle mechanics. PUBLIC HEALTH RELEVANCE: These experiments have two goals: First, by understanding how the stereocilia move together in a mammalian hair cell, we can generate quantitative biophysical models for how hearing works in the kilohertz range. Second, this will help identify the links that permit sliding adhesion of stereocilia. Mutation of similar links is known to cause inherited deafness in mice and humans, and this sort of sliding adhesion represents a new type of mechanism in cell biology.